This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Actin makes different structures throughout cells including a branched actin network at the leading edge of motile cells, parallel actin bundles in filopodia, and actomyosin contractile apparatus during cytokinesis. Tropomyosin participates in many of these structures by controlling access to the actin filament. Much is known about tropomyosin's role in muscle contraction, but much less is understood about its role in nonmuscle cells. It is difficult to study tropomyosin in nonmuscle cells because higher eukaryotes can express up to forty different splice variants from four different genes with up to seven isoforms expressed in any one nonmuscle cell type. This isoform diversity makes it difficult to tease out the contributions of each tropomyosin type. The key to solving the mystery of nonmuscle tropomyosin is finding a simple model organism with a minimal number of nonmuscle tropomyosin isoforms that can be individually studied. Drosophila melanogaster seems to be perfect for this task because only one nonmuscle tropomyosin isoform is known to identify the additional nonmuscle isoform(s) have been inconclusive. We believe the UCSF Mass Spectrometry Facility can help us identify the unknown nonmuscle tropomyosin isoforms. We plan to partially isolate native tropomyosin from Drosophila nonmuscle cells (S2 cells), identify the protein sequence using mass spectrometry, and then characterize the isoforms in vitro an in living S2 cells. These studies will help elucidate the role of tropomyosin isoform diversity in nonmuscle cells.